Investigating the expression of LNCRNAs TMPO-AS1, DDX11-AS1, and POLE gene in head and neck squamous cell carcinoma

Investigating the expression of LNCRNAs TMPO-AS1, DDX11-AS1, and POLE gene in head and neck squamous cell carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Investigating the expression of LNCRNAs TMPO-AS1, DDX11-AS1, and POLE gene in head and neck squamous cell carcinoma Mahnoosh Mokhtarinejad, Maryam Pirhoushiaran Pirhoushiaran, Sara Hesami, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2224676/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background and aim : Head and neck cancer includes all cancers located in the head and neck area, including larynx, oral cavity, nasal cavities, lips, myopic sinuses, tongue and salivary glands. This study was conducted with the aim of evaluating the expression levels of LNCRNAs TMPO-AS1, DDX11-AS1 and POLE mRNA in tumor and normal tissues adjacent to the tumor of patients with HNSCC. Methods and Materials: 50 fresh frozen samples were collected from patients with HNSCC. The expression levels of LNCRNAs TMPO-AS1, DDX11-AS1 and POLE mRNA were measured using real time PCR technique. Results: Based on the findings of this study, an increase in the expression levels of DDX11-AS1 and POLE was observed in tumor tissues compared to the normal tissue adjacent to the tumor (P-value=0.947 and P-value= 0.997). From the point of view of examining the TMPO-AS1 gene, a decrease in expression (P-value=0.163) was observed in tumor samples compared to normal samples adjacent to the tumor. Also, a significant expression correlation (P-value=0.006) was observed between TMPO-AS1 and POLE in normal samples adjacent to the tumor. In addition, a significant expression correlation (P-value=0.001) was observed between DDX11-AS1 and POLE in normal samples adjacent to the tumor. Conclusion: Therefore, DDX11-AS1 and POLE genes probably play a role as oncogenes in HNSCC, while TMPO-AS1 gene is considered as a tumor suppressor gene in this cancer. head and neck squamous cell cancer Long non-coding RNAs TMPO-AS1 DDX11-AS1 POLE Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer worldwide, with almost 600,000 new cases annually ( 1 ). Although many efforts have been made in the field of prevention and treatment of HNSCC in recent years, the number of people with HNSCC is still increasing and the 5-year survival rate is less than 50% ( 2 ). Smoking, excessive alcohol consumption, and human papillomavirus (HPV) infections are the risk factors have been associated with HNSCC developing ( 3 , 4 ). Considering the molecular heterogeneity and diverse etiology of head and neck tumors, a better understanding of molecular alterations as well as the identification of prognostic biomarkers will facilitate the diagnosis and treatment of patients with HNSCC ( 1 , 5 ). RNAs can be classified into two categories: coding RNAs and non-coding RNAs (ncRNAs) ( 6 ). Also, long non-coding RNAs (lncRNAs) (longer than 200 nt) are an important part of ncRNAs. However, the role of lncRNAs in the initiation and progression of HNSCC is poorly understood ( 7 ). Recent studies have shown that lncRNAs can play a regulatory role at the transcriptional, post-transcriptional and epigenetic levels ( 8 ). Accordingly, aberrant lncRNA expression is associated with disruption of cancer pathways including proliferation, migration, and genome stability ( 9 ). Accordingly, with the increasing understanding of lncRNAs, the important value of lncRNAs in the treatment of tumors as prognostic biomarkers for various types of cancer have been recognized ( 10 , 11 ). TMPO-AS1 and DDX11-AS1 can be mentioned among the LncRNAs that have recently attracted the attention of cancer researchers ( 12 , 13 ). DDX11-AS1 is an LncRNA located in the nucleus, whose coding gene is located on chromosome 12p11. In addition, TMPO antisense RNA1 or TMPO-AS1 gene is located on chromosome 12q23. Studies have shown that TMPO-AS1 gene expression has a strong association with the overall survival of patients in various cancers ( 14 , 15 ). The POLE gene plays an essential role in DNA replication and repair of nucleotide errors, which performs this role through its exonuclease domain. This gene located on chromosome 12q24 encodes the catalytic subunit of DNA polymerase epsilon ( 16 ). Since the defect in the repair feature of DNA polymerase epsilon is directly related to the development of cancer; recently, showing germline mutations in POLE exonuclease domains is considered as a biomarker for diagnosing colorectal cancer susceptibility ( 17 , 18 ). Considering the importance and frequency of HNSCC, the selection of diagnostic biomarkers is very important and can play an important role in the early diagnosis of this disease ( 2 ). Accordingly, the aim of this study was to investigate the expression of lncRNAs TMPO-AS1, DDX11-AS1 , and POLE gene in HNSCC patients. 2. Materials And Methods 2.1 Patients and specimens HNSCC tissues and adjacent normal tissues from 50 patients with HNSCC who had undergone tumor removal surgery were collected in Amir Alam and Imam Khomeini Hospital. All of the specimens were checked by a pathologist. All specimens were obtained at the time of surgery and immediately snap-frozen in liquid for the subsequent experiments. This study was approved by the Medical Ethics Committee of Tehran University of Medical Science and informed consent was obtained from all participants. The demographic and clinicopathological data are listed in Table 1. Table.1. the demographic and clinicopathological data of samples. Demographic and Clinicopathological features Frequency (percentage) Age < 40 6 (12) ≥ 40 44 (88) Gender Male 39 (78) Female 11 (22) Smoking Yes 29 (58) No 21 (42) Alcohol consumption Yes 2 (4) No 48 (96) Tumor type Larynx 36 (72) Tongue 10 (20) Palate 4 (8) Tumor Stage II 19 (38) III 31 (62) Tumor degree I 10 (20) II 16 (32) III 14 (28) 2.2 RNA isolation and qRT-PCR analysis For the extraction of total RNA from HNSCC specimens and normal samples, Trizol reagent (Yekta tajhiz, Iran) was used based on the company’s protocol. The DNA complementary to RNA (cDNA) was synthesized from 1 µg of total RNA using the cDNA Synthesis Kit (Yekta tajhiz, Iran), following the manufacturer's instructions. All samples were analyzed in triplicate (three biological replicates) and Succinate Dehydrogenase Complex Flavoprotein Subunit A ( SDHA ) gene was utilized as an endogenous control to confirm the presence and quality of the cDNA template. The RT-PCR assays were carried out for 40 cycles with the successive arrangements: 94°C for 10 min, 60°C for 30 s, and 72°C for 20 s. The data were analyzed and expressed relative to the threshold cycle (CT) values. All qRT-PCR primers used in this study were designed using the Primer 3software tool (Table 2). Table.2. Primers sequences and PCR conditions related to amplification of DDX11-AS1, TMPO-AS1 , POLE , and SDHA . Target Primers (5 , -3 , ) PCR condition Amplicon size DDX11-AS1 Forward :GAA AGT CTG GTT CTG GGA GAT GAC C 95:15min 92:10sec 60:25 sec Cycle:40 132bp Reverse: AGA AAG GTT GCT GGC TGA TGG T TMPO-AS1 Forward: TGA GGT GCT GCA GGA CCG A 95:15min 92:10sec 60:20 sec Cycle:40 69bp Reverse: GGG AGG GGG CTT CGC AGA T POLE Forward: ACG ATT ACT CCA AGC CCA TCC A 95:15min 90:12sec 64:25 sec 50:2min Cycle:40 94bp Reverse: TTC CCA GTG CCA ACG TCC A SDHA Forward: ACG ATT ACT CCA AGC CCA TCC A 95:15min 93:10sec 62:20 sec 20:2min Cycle:40 97bp Reverse: TTC CCA GTG CCA ACG TCC A 2.3 Statistical analysis The statistical assays were performed by SPSS software package (SPSS Inc., Chicago, IL, USA). Student’s t-tests were used for the assays of the differences between the two groups. One way ANOVA is used to compare two qualitative data in three groups. Also, in parametric tests, sample T-test is used to compare two qualitative data in two groups, and Kruskal Wallis test is used to compare two qualitative data in three groups. A value of p < 0.05 was considered statistically significant. 3. Results 3.1 The expression level of DDX11-AS1 We evaluated the differential expression level of the DDX11-AS1 lncRNA between HNSCC tumors and adjacent matched normal tissue. The expression level of DDX11-AS1 in tumor samples has shown an increase compared to the adjacent matched normal tissue, but this increase in expression has not a statistically significant level (P-value = 0.947) (Fig. 1 ). Additionally, DX11-AS1 lncRNA overexpression was not significantly related to any of the clinicopathological features of the patients (P-value > 0.05). 3.2 Comparison of TMPO-AS1 lncRNA expression level in study groups According to the results of this study, TMPO-AS1 lncRNA was expressed at higher levels in tumor samples than in adjacent non-tumor tissues. But, this decrease in expression level was not statistically significant (P-value = 0.163) (Fig. 2 ). Among the clinicopathological features of the patients, the decrease-expression of the TMPO-AS1 lncRNA was only associated with smoking (expression mean in smokers=-1.13, and expression mean in Non-smokers = 0.28). Accordingly, this reduced expression was statistically significant (P-value = 0.05). 3.3 The results of examining POLE gene expression level Examining the expression level of POLE gene showed that the expression of this gene was at higher levels in tumor samples than in adjacent non-tumor tissues. However, this increase in expression level was not statistically significant (P-value = 0.997) (Fig. 3 ). Additionally, POLE gene overexpression was not significantly related to any of the clinicopathological features of the patients (P-value > 0.05). 3.4 Investigating the relationship between DDX11-AS1, TMPO-AS1, and POLE expression in study groups For this purpose, the linear correlation between the expression of DDX11-AS1, TMPO-AS1 , and POLE genes in tumor and normal groups adjacent to the tumor was performed. In tumor samples and normal samples adjacent to the tumor, a weak positive correlation (correlation coefficient = 0.264 and 0.467, respectively) was observed between the expression of DDX11-AS1 and POLE gene. Accordingly, this correlation was statistically significant (P-value = 0.06, and 0.001, respectively) (Fig. 4 ). Additionally, in tumor samples, a weak positive correlation (correlation coefficient = 0.208) was observed between the expression of TMPO-AS1 and POLE gene. Accordingly, this correlation was not statistically significant (P-value = 0.148). On the other hand, in normal samples adjacent to the tumor, a moderate positive correlation (correlation coefficient = 0.383) was observed between the expression of TMPO-AS1 and POLE gene and this correlation was statistically significant (P-value = 0.06) (Fig. 5 ). 4. Discussion HNSCC is one of the most common cancers. Among the salient features of this disease is the low overall survival rate of HNSCC patients despite significant advances in clinical research and new treatments ( 19 ). Accordingly, patients with the same clinical and pathological stages may show different prognosis. Recently, molecular biomarkers have shown promise in their prognostic value and have attracted increasing attention ( 20 ). Many studies have shown that mRNAs and lncRNAs can act as molecular markers to predict the outcome of HNSCC ( 21 ). Accordingly, in this study, we investigated the expression of DDX11-AS1, TMPO-AS1 , and POLE gene in HNSCC due to the importance of each of the mentioned genes in the initiation and progression of cancers. Previous studies have reported several lncRNAs, such as HOTAIR and H19, are abnormally overexpressed and associated with tumor aggressive behavior and unfavorable prognosis in HNSCC. However, the expression patterns of DDX11-AS1, TMPO-AS1 , and POLE genes in human cancer remain largely unexplored ( 22 ). Here, we measured the expression level of DDX11-AS1 in HNSCC specimens by qRT-PCR and found that its expression was higher in a large fraction of fresh HNSCC tissue compared with its paired adjacent nontumor tissue. But, this expression difference was not statistically significant. Shi et al. (2017) showed that DDX11-AS1 plays a role in DNA repair, metabolism, and cell cycle ( 23 ). On the other hand, Li et al. (2019) also showed that DDX11-AS1 exerts its oncogenic nature by suppressing Large Tumor Suppressor Kinase 2 ( LATS2 ) gene expression, thereby increasing cell proliferation and cancer progression ( 24 ). In another study conducted by Marches et al. (2016) on colorectal cancer, they showed that the decrease in the expression of this gene leads to a decrease in cell proliferation, and arrest of the cell cycle in the G0/G1 cycle. According to all the mentioned studies and the results of this study based on increasing the expression level of this gene in tumor samples compared to normal samples, therefore, this lncRNA can be used as a potential therapeutic target in cancers ( 25 ). Other genes examined in this study were TMPO-AS1 and POLE . Based on the results obtained from this research, TMPO-AS1 expression was not increased in tumor samples compared to normal samples. Accordingly, Yang et al. (2019) investigated the role of TMPO-AS1 in cervical cancer. The results of this study showed that the increase in the expression of TMPO-AS1 leads to a decrease in the expression level of miR 577 and as a result, an increase in the expression level of the RAB14 gene (a member of the RAS oncogene family). Finally, increasing the expression of this gene plays an important role in the development of cancer and tumorigenesis ( 26 ). Additionally, Huang et al. (2018) showed in their study that TMPO-AS1 can induce cell proliferation in prostate cancer through the effect on cell cycle and apoptosis process ( 27 ). According to the results obtained between the expression of TMPO-AS1, DDX11-AS1 , and POLE , a positive linear correlation was observed in normal samples adjacent to the tumor, which confirms the results of other studies regarding the role of these genes in similar pathways. Contrary to this, the absence of a positive linear correlation between TMPO-AS1, DDX11-AS1 , and POLE mRNA in tumor samples may be due to the change in gene expression pattern by mutations, epigenetic changes or disruption in the regulatory factors of each. In conclusion, in this study, no significant difference was observed between the expression of DDX1-AS1, TMPO-AS1 , and POLE gene in tumor samples and samples adjacent to the tumor. Also, a significant positive correlation between DDX11-AS1 , TMPO-AS1 , and POLE gene expression was observed only in normal samples adjacent to the tumor. In this study, we showed that the investigated genetic factors such as lncRNA can play an important role as biomarkers in cancer diagnosis. Based on this, the evaluation of information related to HNSCC can lead to its initial screening and as a result reduce the death rate resulting from it. However, more studies are needed to prove this information. Declarations Conflict of interest There are no conflicts of interest. Compliance with Ethical Standards This study was funded by Tehran University of Medical Science (grant number IR.TUMS.MEDICINE.REC.1398.550). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. References Johnson DE, Burtness B, Leemans CR, Lui VWY, Bauman JE, Grandis JR (2020) Head and neck squamous cell carcinoma. Nat reviews Disease primers 6(1):1–22 Economopoulou P, De Bree R, Kotsantis I, Psyrri A (2019) Diagnostic tumor markers in head and neck squamous cell carcinoma (HNSCC) in the clinical setting. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2224676","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":150437125,"identity":"3f09ee11-3f32-4d7a-81f3-abe875512364","order_by":0,"name":"Mahnoosh Mokhtarinejad","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mahnoosh","middleName":"","lastName":"Mokhtarinejad","suffix":""},{"id":150437126,"identity":"a369534f-1aed-4cfa-bef1-1cabab5859a9","order_by":1,"name":"Maryam Pirhoushiaran Pirhoushiaran","email":"","orcid":"","institution":"Tehran 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15:51:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":51795,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation of \u003cem\u003eTPMO-AS1\u003c/em\u003eand \u003cem\u003ePOLE\u003c/em\u003e gene expression between tumor tissues and normal tissues adjacent to the tumor.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2224676/v1/ba7de9d1cf2d6afb29dd3892.png"},{"id":30525227,"identity":"45027406-fdea-48ed-9c7c-b61c67a44cb3","added_by":"auto","created_at":"2022-12-19 16:05:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":476740,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2224676/v1/5146f27a-6ac8-4196-9069-d145ec0d632e.pdf"}],"financialInterests":"","formattedTitle":"Investigating the expression of LNCRNAs TMPO-AS1, DDX11-AS1, and POLE gene in head and neck squamous cell carcinoma","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHead and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer worldwide, with almost 600,000 new cases annually (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Although many efforts have been made in the field of prevention and treatment of HNSCC in recent years, the number of people with HNSCC is still increasing and the 5-year survival rate is less than 50% (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Smoking, excessive alcohol consumption, and human papillomavirus (HPV) infections are the risk factors have been associated with HNSCC developing (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Considering the molecular heterogeneity and diverse etiology of head and neck tumors, a better understanding of molecular alterations as well as the identification of prognostic biomarkers will facilitate the diagnosis and treatment of patients with HNSCC (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRNAs can be classified into two categories: coding RNAs and non-coding RNAs (ncRNAs) (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Also, long non-coding RNAs (lncRNAs) (longer than 200 nt) are an important part of ncRNAs. However, the role of lncRNAs in the initiation and progression of HNSCC is poorly understood (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Recent studies have shown that lncRNAs can play a regulatory role at the transcriptional, post-transcriptional and epigenetic levels (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Accordingly, aberrant lncRNA expression is associated with disruption of cancer pathways including proliferation, migration, and genome stability (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Accordingly, with the increasing understanding of lncRNAs, the important value of lncRNAs in the treatment of tumors as prognostic biomarkers for various types of cancer have been recognized (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eTMPO-AS1\u003c/em\u003e and \u003cem\u003eDDX11-AS1\u003c/em\u003e can be mentioned among the LncRNAs that have recently attracted the attention of cancer researchers (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). DDX11-AS1 is an LncRNA located in the nucleus, whose coding gene is located on chromosome 12p11. In addition, TMPO antisense RNA1 or \u003cem\u003eTMPO-AS1\u003c/em\u003e gene is located on chromosome 12q23. Studies have shown that \u003cem\u003eTMPO-AS1\u003c/em\u003e gene expression has a strong association with the overall survival of patients in various cancers (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ePOLE\u003c/em\u003e gene plays an essential role in DNA replication and repair of nucleotide errors, which performs this role through its exonuclease domain. This gene located on chromosome 12q24 encodes the catalytic subunit of DNA polymerase epsilon (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Since the defect in the repair feature of DNA polymerase epsilon is directly related to the development of cancer; recently, showing germline mutations in POLE exonuclease domains is considered as a biomarker for diagnosing colorectal cancer susceptibility (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConsidering the importance and frequency of HNSCC, the selection of diagnostic biomarkers is very important and can play an important role in the early diagnosis of this disease (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Accordingly, the aim of this study was to investigate the expression of lncRNAs \u003cem\u003eTMPO-AS1, DDX11-AS1\u003c/em\u003e, and \u003cem\u003ePOLE\u003c/em\u003e gene in HNSCC patients.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Patients and specimens\u003c/h2\u003e \u003cp\u003eHNSCC tissues and adjacent normal tissues from 50 patients with HNSCC who had undergone tumor removal surgery were collected in Amir Alam and Imam Khomeini Hospital. All of the specimens were checked by a pathologist. All specimens were obtained at the time of surgery and immediately snap-frozen in liquid for the subsequent experiments. This study was approved by the Medical Ethics Committee of Tehran University of Medical Science and informed consent was obtained from all participants. The demographic and clinicopathological data are listed in Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable.1.\u003c/b\u003e the demographic and clinicopathological data of samples.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eDemographic and Clinicopathological features\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFrequency (percentage)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eAge\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (12)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44 (88)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eGender\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39 (78)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 (22)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eSmoking\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29 (58)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21 (42)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eAlcohol consumption\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48 (96)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eTumor type\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLarynx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36 (72)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTongue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (20)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePalate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eTumor Stage\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19 (38)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31 (62)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eTumor degree\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (20)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16 (32)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14 (28)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 RNA isolation and qRT-PCR analysis\u003c/h2\u003e \u003cp\u003eFor the extraction of total RNA from HNSCC specimens and normal samples, Trizol reagent (Yekta tajhiz, Iran) was used based on the company\u0026rsquo;s protocol. The DNA complementary to RNA (cDNA) was synthesized from 1 \u0026micro;g of total RNA using the cDNA Synthesis Kit (Yekta tajhiz, Iran), following the manufacturer's instructions. All samples were analyzed in triplicate (three biological replicates) and Succinate Dehydrogenase Complex Flavoprotein Subunit A (\u003cem\u003eSDHA\u003c/em\u003e) gene was utilized as an endogenous control to confirm the presence and quality of the cDNA template. The RT-PCR assays were carried out for 40 cycles with the successive arrangements: 94\u0026deg;C for 10 min, 60\u0026deg;C for 30 s, and 72\u0026deg;C for 20 s. The data were analyzed and expressed relative to the threshold cycle (CT) values. All qRT-PCR primers used in this study were designed using the Primer 3software tool (Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable.2.\u003c/b\u003e Primers sequences and PCR conditions related to amplification of \u003cem\u003eDDX11-AS1, TMPO-AS1\u003c/em\u003e, \u003cem\u003ePOLE\u003c/em\u003e, and \u003cem\u003eSDHA\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTarget\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimers (5\u003csup\u003e,\u003c/sup\u003e-3\u003csup\u003e,\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePCR condition\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAmplicon size\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eDDX11-AS1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward :GAA AGT CTG GTT CTG GGA GAT GAC C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e95:15min\u003c/p\u003e \u003cp\u003e92:10sec\u003c/p\u003e \u003cp\u003e60:25 sec\u003c/p\u003e \u003cp\u003eCycle:40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e132bp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse: AGA AAG GTT GCT GGC TGA TGG T\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eTMPO-AS1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward: TGA GGT GCT GCA GGA CCG A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e95:15min\u003c/p\u003e \u003cp\u003e92:10sec\u003c/p\u003e \u003cp\u003e60:20 sec\u003c/p\u003e \u003cp\u003eCycle:40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e69bp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse: GGG AGG GGG CTT CGC AGA T\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003ePOLE\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward: ACG ATT ACT CCA AGC CCA TCC A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e95:15min\u003c/p\u003e \u003cp\u003e90:12sec\u003c/p\u003e \u003cp\u003e64:25 sec\u003c/p\u003e \u003cp\u003e50:2min\u003c/p\u003e \u003cp\u003eCycle:40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e94bp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse: TTC CCA GTG CCA ACG TCC A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eSDHA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward: ACG ATT ACT CCA AGC CCA TCC A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e95:15min\u003c/p\u003e \u003cp\u003e93:10sec\u003c/p\u003e \u003cp\u003e62:20 sec\u003c/p\u003e \u003cp\u003e20:2min\u003c/p\u003e \u003cp\u003eCycle:40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e97bp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse: TTC CCA GTG CCA ACG TCC A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical assays were performed by SPSS software package (SPSS Inc., Chicago, IL, USA). Student\u0026rsquo;s t-tests were used for the assays of the differences between the two groups. One way ANOVA is used to compare two qualitative data in three groups. Also, in parametric tests, sample T-test is used to compare two qualitative data in two groups, and Kruskal Wallis test is used to compare two qualitative data in three groups. A value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1 The expression level of DDX11-AS1\u003c/h2\u003e \u003cp\u003eWe evaluated the differential expression level of the \u003cem\u003eDDX11-AS1\u003c/em\u003e lncRNA between HNSCC tumors and adjacent matched normal tissue. The expression level of \u003cem\u003eDDX11-AS1\u003c/em\u003e in tumor samples has shown an increase compared to the adjacent matched normal tissue, but this increase in expression has not a statistically significant level (P-value\u0026thinsp;=\u0026thinsp;0.947) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Additionally, \u003cem\u003eDX11-AS1\u003c/em\u003e lncRNA overexpression was not significantly related to any of the clinicopathological features of the patients (P-value\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Comparison of \u003cem\u003eTMPO-AS1\u003c/em\u003e lncRNA expression level in study groups\u003c/h2\u003e \u003cp\u003eAccording to the results of this study, \u003cem\u003eTMPO-AS1\u003c/em\u003e lncRNA was expressed at higher levels in tumor samples than in adjacent non-tumor tissues. But, this decrease in expression level was not statistically significant (P-value\u0026thinsp;=\u0026thinsp;0.163) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Among the clinicopathological features of the patients, the decrease-expression of the \u003cem\u003eTMPO-AS1\u003c/em\u003e lncRNA was only associated with smoking (expression mean in smokers=-1.13, and expression mean in Non-smokers\u0026thinsp;=\u0026thinsp;0.28). Accordingly, this reduced expression was statistically significant (P-value\u0026thinsp;=\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.3 The results of examining \u003cem\u003ePOLE\u003c/em\u003e gene expression level\u003c/h2\u003e \u003cp\u003eExamining the expression level of \u003cem\u003ePOLE\u003c/em\u003e gene showed that the expression of this gene was at higher levels in tumor samples than in adjacent non-tumor tissues. However, this increase in expression level was not statistically significant (P-value\u0026thinsp;=\u0026thinsp;0.997) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Additionally, \u003cem\u003ePOLE\u003c/em\u003e gene overexpression was not significantly related to any of the clinicopathological features of the patients (P-value\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Investigating the relationship between DDX11-AS1, TMPO-AS1, and \u003cem\u003ePOLE\u003c/em\u003e expression in study groups\u003c/h2\u003e \u003cp\u003eFor this purpose, the linear correlation between the expression of \u003cem\u003eDDX11-AS1, TMPO-AS1\u003c/em\u003e, and \u003cem\u003ePOLE\u003c/em\u003e genes in tumor and normal groups adjacent to the tumor was performed. In tumor samples and normal samples adjacent to the tumor, a weak positive correlation (correlation coefficient\u0026thinsp;=\u0026thinsp;0.264 and 0.467, respectively) was observed between the expression of \u003cem\u003eDDX11-AS1\u003c/em\u003e and \u003cem\u003ePOLE\u003c/em\u003e gene. Accordingly, this correlation was statistically significant (P-value\u0026thinsp;=\u0026thinsp;0.06, and 0.001, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Additionally, in tumor samples, a weak positive correlation (correlation coefficient\u0026thinsp;=\u0026thinsp;0.208) was observed between the expression of \u003cem\u003eTMPO-AS1\u003c/em\u003e and \u003cem\u003ePOLE\u003c/em\u003e gene. Accordingly, this correlation was not statistically significant (P-value\u0026thinsp;=\u0026thinsp;0.148). On the other hand, in normal samples adjacent to the tumor, a moderate positive correlation (correlation coefficient\u0026thinsp;=\u0026thinsp;0.383) was observed between the expression of \u003cem\u003eTMPO-AS1\u003c/em\u003e and \u003cem\u003ePOLE\u003c/em\u003e gene and this correlation was statistically significant (P-value\u0026thinsp;=\u0026thinsp;0.06) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eHNSCC is one of the most common cancers. Among the salient features of this disease is the low overall survival rate of HNSCC patients despite significant advances in clinical research and new treatments (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Accordingly, patients with the same clinical and pathological stages may show different prognosis. Recently, molecular biomarkers have shown promise in their prognostic value and have attracted increasing attention (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Many studies have shown that mRNAs and lncRNAs can act as molecular markers to predict the outcome of HNSCC (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Accordingly, in this study, we investigated the expression of \u003cem\u003eDDX11-AS1, TMPO-AS1\u003c/em\u003e, and \u003cem\u003ePOLE\u003c/em\u003e gene in HNSCC due to the importance of each of the mentioned genes in the initiation and progression of cancers.\u003c/p\u003e \u003cp\u003ePrevious studies have reported several lncRNAs, such as HOTAIR and H19, are abnormally overexpressed and associated with tumor aggressive behavior and unfavorable prognosis in HNSCC. However, the expression patterns of \u003cem\u003eDDX11-AS1, TMPO-AS1\u003c/em\u003e, and \u003cem\u003ePOLE\u003c/em\u003e genes in human cancer remain largely unexplored (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHere, we measured the expression level of \u003cem\u003eDDX11-AS1\u003c/em\u003e in HNSCC specimens by qRT-PCR and found that its expression was higher in a large fraction of fresh HNSCC tissue compared with its paired adjacent nontumor tissue. But, this expression difference was not statistically significant. Shi et al. (2017) showed that \u003cem\u003eDDX11-AS1\u003c/em\u003e plays a role in DNA repair, metabolism, and cell cycle (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). On the other hand, Li et al. (2019) also showed that \u003cem\u003eDDX11-AS1\u003c/em\u003e exerts its oncogenic nature by suppressing Large Tumor Suppressor Kinase 2 (\u003cem\u003eLATS2\u003c/em\u003e) gene expression, thereby increasing cell proliferation and cancer progression (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). In another study conducted by Marches et al. (2016) on colorectal cancer, they showed that the decrease in the expression of this gene leads to a decrease in cell proliferation, and arrest of the cell cycle in the G0/G1 cycle. According to all the mentioned studies and the results of this study based on increasing the expression level of this gene in tumor samples compared to normal samples, therefore, this lncRNA can be used as a potential therapeutic target in cancers (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOther genes examined in this study were \u003cem\u003eTMPO-AS1\u003c/em\u003e and \u003cem\u003ePOLE\u003c/em\u003e. Based on the results obtained from this research, \u003cem\u003eTMPO-AS1\u003c/em\u003e expression was not increased in tumor samples compared to normal samples. Accordingly, Yang et al. (2019) investigated the role of \u003cem\u003eTMPO-AS1\u003c/em\u003e in cervical cancer. The results of this study showed that the increase in the expression of \u003cem\u003eTMPO-AS1\u003c/em\u003e leads to a decrease in the expression level of miR 577 and as a result, an increase in the expression level of the RAB14 gene (a member of the RAS oncogene family). Finally, increasing the expression of this gene plays an important role in the development of cancer and tumorigenesis (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Additionally, Huang et al. (2018) showed in their study that \u003cem\u003eTMPO-AS1\u003c/em\u003e can induce cell proliferation in prostate cancer through the effect on cell cycle and apoptosis process (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to the results obtained between the expression of \u003cem\u003eTMPO-AS1, DDX11-AS1\u003c/em\u003e, and \u003cem\u003ePOLE\u003c/em\u003e, a positive linear correlation was observed in normal samples adjacent to the tumor, which confirms the results of other studies regarding the role of these genes in similar pathways. Contrary to this, the absence of a positive linear correlation between \u003cem\u003eTMPO-AS1, DDX11-AS1\u003c/em\u003e, and \u003cem\u003ePOLE\u003c/em\u003e mRNA in tumor samples may be due to the change in gene expression pattern by mutations, epigenetic changes or disruption in the regulatory factors of each.\u003c/p\u003e \u003cp\u003eIn conclusion, in this study, no significant difference was observed between the expression of \u003cem\u003eDDX1-AS1, TMPO-AS1\u003c/em\u003e, and \u003cem\u003ePOLE\u003c/em\u003e gene in tumor samples and samples adjacent to the tumor. Also, a significant positive correlation between \u003cem\u003eDDX11-AS1\u003c/em\u003e, \u003cem\u003eTMPO-AS1\u003c/em\u003e, and \u003cem\u003ePOLE\u003c/em\u003e gene expression was observed only in normal samples adjacent to the tumor. In this study, we showed that the investigated genetic factors such as lncRNA can play an important role as biomarkers in cancer diagnosis. Based on this, the evaluation of information related to HNSCC can lead to its initial screening and as a result reduce the death rate resulting from it. However, more studies are needed to prove this information.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by Tehran University of Medical Science (grant number IR.TUMS.MEDICINE.REC.1398.550). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJohnson DE, Burtness B, Leemans CR, Lui VWY, Bauman JE, Grandis JR (2020) Head and neck squamous cell carcinoma. Nat reviews Disease primers 6(1):1\u0026ndash;22\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEconomopoulou P, De Bree R, Kotsantis I, Psyrri A (2019) Diagnostic tumor markers in head and neck squamous cell carcinoma (HNSCC) in the clinical setting. Front Oncol 9:827\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGildener-Leapman N, Ferris RL, Bauman JE (2013) Promising systemic immunotherapies in head and neck squamous cell carcinoma. 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Cell Death Discovery 7(1):1\u0026ndash;18\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Wang H, Yang W, Qian Y (2020) Prediction of Specific Subtypes and Common Markers of Non-Small Cell Lung Cancer Based on Competing Endogenous RNA Network. Med Sci Monitor: Int Med J Experimental Clin Res 26:e922280\u0026ndash;e922281\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuerra J, Pinto C, Pinto D, Pinheiro M, Silva R, Peixoto A et al (2017) POLE somatic mutations in advanced colorectal cancer. Cancer Med 6(12):2966\u0026ndash;2971\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuenther M, Veninga V, Kumbrink J, Haas M, Westphalen CB, Kruger S et al (2018) POLE gene hotspot mutations in advanced pancreatic cancer. J Cancer Res Clin Oncol 144(11):2161\u0026ndash;2166\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL\u0026oacute;pez-Verd\u0026iacute;n S, Lavalle-Carrasco J, Carre\u0026oacute;n-Burciaga RG, Seraf\u0026iacute;n-Higuera N, Molina-Frechero N, Gonz\u0026aacute;lez-Gonz\u0026aacute;lez R et al (2018) Molecular markers of anticancer drug resistance in head and neck squamous cell carcinoma: a literature review. Cancers 10(10):376\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuzaffar J, Bari S, Kirtane K, Chung CH (2021) Recent advances and future directions in clinical management of head and neck squamous cell carcinoma. Cancers 13(2):338\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGavrielatou N, Doumas S, Economopoulou P, Foukas PG, Psyrri A (2020) Biomarkers for immunotherapy response in head and neck cancer. Cancer Treat Rev 84:101977\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuglas K, Bogaczyńska M, Kolenda T, Ryś M, Teresiak A, Bliźniak R et al (2017) lncRNA in HNSCC: challenges and potential. Contemp Oncology/Wsp\u0026oacute;łczesna Onkologia 21(4):259\u0026ndash;266\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Wang S, Ren Y, Zhou X (2020) The role of lncRNA crosstalk in leading cancer metastasis of head and neck squamous cell carcinoma. Front Oncol 10:561833\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi M, Zhang X-Y, Yu H, Xiang S-H, Xu L, Wei J et al (2017) DDX11-AS1 as potential therapy targets for human hepatocellular carcinoma. Oncotarget 8(27):44195\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Zhuang W, Huang M, Li X (2019) Long noncoding RNA DDX11-AS1 epigenetically represses LATS2 by interacting with EZH2 and DNMT1 in hepatocellular carcinoma. Biochem Biophys Res Commun 514(4):1051\u0026ndash;1057\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarchese FP, Grossi E, Mar\u0026iacute;n-B\u0026eacute;jar O, Bharti SK, Raimondi I, Gonz\u0026aacute;lez J et al (2016) A long noncoding RNA regulates sister chromatid cohesion. Mol Cell 63(3):397\u0026ndash;407\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang J, Liang B, Hou S (2019) TMPO-AS1 promotes cervical cancer progression by upregulating RAB14 via sponging miR‐577. J Gene Med 21(11):e3125\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang W, Su X, Yan W, Kong Z, Wang D, Huang Y et al (2018) Overexpression of AR-regulated lncRNA TMPO‐AS1 correlates with tumor progression and poor prognosis in prostate cancer. Prostate 78(16):1248\u0026ndash;1261\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"head and neck squamous cell cancer, Long non-coding RNAs, TMPO-AS1, DDX11-AS1, POLE","lastPublishedDoi":"10.21203/rs.3.rs-2224676/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2224676/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground and aim\u003c/strong\u003e: Head and neck cancer includes all cancers located in the head and neck area, including larynx, oral cavity, nasal cavities, lips, myopic sinuses, tongue and salivary glands. This study was conducted with the aim of evaluating the expression levels of LNCRNAs TMPO-AS1, DDX11-AS1 and POLE mRNA in tumor and normal tissues adjacent to the tumor of patients with HNSCC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods and Materials:\u003c/strong\u003e 50 fresh frozen samples were collected from patients with HNSCC. The expression levels of LNCRNAs TMPO-AS1, DDX11-AS1 and POLE mRNA were measured using real time PCR technique.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003eBased on the findings of this study, an increase in the expression levels of DDX11-AS1 and POLE was observed in tumor tissues compared to the normal tissue adjacent to the tumor (P-value=0.947 and P-value= 0.997). From the point of view of examining the TMPO-AS1 gene, a decrease in expression (P-value=0.163) was observed in tumor samples compared to normal samples adjacent to the tumor. Also, a significant expression correlation (P-value=0.006) was observed between TMPO-AS1 and POLE in normal samples adjacent to the tumor. In addition, a significant expression correlation (P-value=0.001) was observed between DDX11-AS1 and POLE in normal samples adjacent to the tumor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003eTherefore, DDX11-AS1 and POLE genes probably play a role as oncogenes in HNSCC, while TMPO-AS1 gene is considered as a tumor suppressor gene in this cancer.\u003c/p\u003e","manuscriptTitle":"Investigating the expression of LNCRNAs TMPO-AS1, DDX11-AS1, and POLE gene in head and neck squamous cell carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-10 15:51:40","doi":"10.21203/rs.3.rs-2224676/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"df873bec-850c-4922-b0f1-0df41f4deff7","owner":[],"postedDate":"November 10th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-12-19T16:05:33+00:00","versionOfRecord":[],"versionCreatedAt":"2022-11-10 15:51:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2224676","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2224676","identity":"rs-2224676","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}